Disruption of lipid order by short-chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcϵRI Available to Purchase

Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, and their liquid-ordered properties appear to be important for their function. We investigated the possibility of using amphiphiles to disrupt lipid rafts and thereby inhibit IgE-FcϵRI signaling. We find that short-chain ceramides –C₂-ceramide and C₆-ceramide – decrease plasma membrane lipid order and reduce the extent of fluorescence resonance energy transfer between lipid-raft-associated molecules on intact cells; by contrast,biologically inactive C₂-dihydroceramide does neither. Structural perturbations by these ceramides parallel their inhibitory effects on antigen-stimulated Ca²⁺ mobilization in RBL mast cells in the presence and absence of extracellular Ca²⁺. Similar inhibition of Ca²⁺ mobilization is caused by n-butanol, which prevents phosphatidic acid production by phospholipase D, but not by t-butanol, which does not prevent phosphatidic acid production. These results and previously reported effects of short-chain ceramides on phospholipase D activity prompted us to compare the effects of C₂-ceramide,C₂-dihydroceramide and C₁₆-ceramide on phospholipase D1 and phospholipase D2 activities in vitro. We find that the effects of these ceramides on phospholipase D1 activity strongly correlate with their effects on antigen-stimulated Ca²⁺ mobilization and with their disruption of lipid order. Our results indicate that phospholipase D activity is upstream of antigen-stimulated Ca²⁺ mobilization in these cells, and they demonstrate that ceramides can serve as useful probes for investigating roles of plasma membrane structure and phospholipase D activity in cellular signaling.